Prevalence and methodologies for detection,characterization and subtyping of Listeria monocytogenes and L.ivanovii in foods and environmental sources

Jin-Qing ChenPtrick RegnPongpn LksnlmiStephnie HeleyZonglin Hu

aWinchester Engineering&Analytical Center(WEAC),HFR-NE460,Office of Regulatory Affairs,US Food and Drug Administration,109 Holton Street,Winchester,MA 01890,USA

bMD DHMH Laboratories Administration,Division of Microbiology,Room 528,1770 Ashland Ave.,Baltimore,MD 21205,USA

Abstract

Keywords:Foodborne pathogens;Listeria monocytogenes;Listeria ivanovii;Molecular characterization;Subtyping

1.Introduction

Listeria monocytogenes(L.monocytogenes)is one of the most important foodborne pathogens.Its infection can cause listeriosis,a lethal disease for humans,accounting for approximately 19% of total death caused by all the major known foodborne pathogens representing a significant medical burden and public health threat[1,2].L.monocytogenesis widely distributed in various foods and environmental sources[3].During the past decades,a variety of methodologies have been developed for detection,characterization and subtyping ofL.monocytogenesin foods and environmental sources.This review comprehensively described multiple methodologies that can be used,in combination with PCR-based methodologies(for review see Chen et al.),to detect,characterize and subtypeL.monocytogenesandL.ivanoviistrains present in foods and environmental sources according to particular situations and needs.

1.1.Listeria monocytogenes,L.ivanovii and world-wide incidence of listeriosis

The species in genusListeriaare small gram-positive,rod-shaped,facultatively anaerobic,and non-spore-forming bacteria.L.monocytogenes,L.ivanovii,L.seeligeri,L.innocua,L.welshimeri,L.grayi,and L.marthii,are typically included in the genusListeria[4,5].In addition,several newListerialike species have been reported[6–10].To date,a total of 17ListeriaandListeria-like species have been identified[11].Among them,L.monocytogenesandL.ivanoviiare pathogenic[4,5].

Listeriosis caused byL.monocytogenesincludes invasive listeriosis with meningitis,septicemia,primary bacteremia,endocarditis,non-meningitic central nervous system(CNS)infection,influenza-like illness,conjunctivitis,and severe noninvasive listeriosis with febrile gastroenteritis in susceptible individuals,including pregnant women,newborns,elderly,immunocompromised persons and those infected with human immunodeficiency virus and patients with organ transplant or with various types of cancer[2,5,12–20].L.monocytogenesis capable of migrating from pregnant mother to fetus in utero,potentially leading to stillbirth or abortion.The invasive form of listeriosis is a severe disease with high hospitalization rate(90%)and fatality rate(20%–30%)[21].L.monocytogenesis also responsible for mild flulike illness or asymptomatic infection among general health populations.

Since 1980,numerous listeriosis outbreaks in humans have occurred[12,14,22–49],including those occurred in Italy[30],Germany[34,35],Sweden[36],Portugal[37],Netherland[38],France[39–41],England/Wales[42],Spain[32],USA[50]and Canada[48],China[51–56],India[57],Japan[58],Thailand[59],South America[60,61],Australia-New Zealand[62,63]and African counties[49].It was estimated that approximately 1650 cases of listeriosis occurred in Europe in 2009[64]and 1600 listeriosis cases occurred annually in the United States,leading to 260 deaths[17,65]);approximately 135–201 annual listeriosis cases in Japan[58];and that the cost of listeriosis,including premature death,medical expenses and decreased productivity,was as high as$2.6 billion/year just in the USA alone[66].In developing countries,L.monocytogenesis responsible for the highest hospitalization rates amongst the known foodborne pathogens linked to both sporadic episodes and large outbreaks of human illness worldwide,representing one of the most important causes of death related to foodborne infection[67].Furthermore,L.monocytogenesoutbreaks have also been reported in animals such as birds[68],poultry[69–71],livestock animals and ruminants,resulting in economic loss[72–75].

L.ivanoviiis also widely distributed in nature,frequently found in warm-blooded animals and has been isolated in fecal specimens from healthy hosts,soil,water,waste,vegetables,processed silage,foods and food-processing plants[76].WhileL.ivanoviiis generally considered to infect mainly ruminants[4],several cases ofL.ivanovii–associated gastroenteritis and bacteremia in human have also been reported[77–80].Listeriosis caused byL.ivanoviiis an important problem in ruminants,as it has caused epidemic abortion,stillbirths and encephalitis in ruminants[81–85].

Listeriosis is predominantly the foodborne disease since 99% of all human listeriosis cases are resulted from consumption of food products contaminated withL.monocytogenes,although it can be transmitted from person to person or via utero/parental transmission[44,47,65].Importantly,about 10–100 CFU ofL.monocytogenesare capable of causing listeriosis,although infective doses required for causing listeriosis mainly depend on strains and susceptibility of the individuals[21,86].

L.monocytogenesandL.ivanoviipossess unique capabilities of invading host cells,replicating in host cell cytosol after phagosomal escape,and spreading from cell to cell.These capabilities are attributed correspondingly to theirinlABinternalization locus,Listeriapathogenicity island-1(LIPI-1),andhptintracellular growth locus,respectively[4,87].InlABinternalization locus encodes two surface proteins,namelyinlAandInlB.Via their Leucine-Rich Repeat(LRR)domains,bothinlAandinlBbind their corresponding receptors on host cell surface,i.e.InlA binds human E-cadherin[88,89]while InlB binds hepatocyte growth factor receptor Met[90],respectively.Their combined actions mediate bacterial internalization and invasion into host cells. LIPI-1, the central virulence gene cluster,encodes a pore-forming toxin listeriolysin O(LLO)and two phospholipases C(PlcA and PlcB),which cooperate to lyse the phagocytic vacuole membrane of host cells[4,91].Thehptintracellular growth locus encodes hexose-6-phosphate transporter and actin assembly-inducing protein(ActA),which play an important role in intracellular bacterial growth,cell-to-cell spread and actin polymerization[4].The virulence ofL.monocytogenesis mainly regulated by six genes(PrfA,PI-PLC,LLO,Mpl,ActA and PC-PLC)residing inPrfA-dependent virulence gene cluster and other virulence-related genes(e.g.internalin genes)located outside this gene cluster[4].Furthermore,L.monocytogenescarries a gene cluster of five genes termed stress survival islet 1(SSI-1),contributing to the survival of cells under suboptimal conditions,including low pH and high salt concentrations,in food environments[92].

2.Serotypes and prevalence of various species of L.monocytogenes and Listeria spp.in foods and environmental sources

2.1.A brief introduction to serotypes of L.monocytogenes strains

Listeriaspecies possess two types of group-specific surface proteins,i.e.somatic(O)and flagellar(H)antigens.A total of 15 subtypes ofListeriaO-antigens(namely I–XV)and 4 subtypes(namely A-D)of H-antigens have been known.Both O-and H-subtypes of antigens have been used as targets for serological detection with corresponding antibodies.Serotypes of individualListeriastrains can be determined by unique combinations of their O-and H-antigens(Table 1).Fifteen serotypes(1/2a,1/2b,1/2c,3a,3b,3c,4a,4b,4c,4d,4e 5,6a,6b and 7)have been recognized inL.monocytogenesstrains based on group-specificListeriaO-and H-antigens in slide agglutination[93,94].Theyare mainly classified into Division I(1/2b,3b,4b,4d,and 4e),Division II(1/2a,1/2c,3a,and3c);Division III serotypes(4a and 4c)[95],and Division IV(4a,4c and atypical 4b),which are less frequently found[3,20].Of which,L.monocytogenesserotypes 1/2a,1/2b and 4b account for 98% of total isolates of the documented human listeriosis cases and the isolates derived from environmental and food sources,while serotypes 4a and 4c are rarely associated with outbreaks of the disease[12,56,93,96–98].Serotype 4b strains are mainly responsible for most of the epidemic listeriosis while serotypes 1/2a and 1/2b are mainly linked to sporadic infection ofL.monocytogenes[99].

Table 1 Listeria Serotypes based on somatic(O)and flagellar(H)antigens.a

2.2.Prevalence of various L.monocytogenes strains and listeria spp.in foods and environmental sources

The opportunisticL.monocytogenesstrains possess unique capabilities enabling them to survive and multiply under normal and severe environmental conditions and to increase their widespread prevalence in different foods and environmental sources:(A)unlike other pathogenic foodborne bacteria,e.g.E coli0157:H7 andSalmonellawhose proliferation is generally inhibited at cold(refrigerated)temperatures,this psychotropic organism is capable of surviving and/or even thriving at such temperatures,providing a unique advantage over other bacteria in this stressful environment.Extension of refrigerated storage of ready-to-eat(RTE)foods allowsL.monocytogenesstrains to grow in contaminated foods during production chain,subsequent handling,processing processes and post-processing storage.For those refrigerated products that have a long shelflife andL.monocytogenes-contaminated foods that are generally eatable without or with little prior heating, they can impose much greater threat[100];(B)L.monocytogenesstrains are capable of surviving and even multiplying in an acidic or alkaline pH ranging 4.0–9.6[21,101,102],a broad temperature range(?0.4 to 50℃),high salt conditions, oxidative stress, osmotic stress,and carbon starvation etc.They are also capable of being resistant to UV radiation,γ-rays and X-rays[21,101–104],possessing a great adaptability to such stressful environment conditions;(C)more importantly,L.monocytogenesstrains are capable of readily producing biofilm,an assemblage of surface-associated microbial cells enclosed in an extracellular polymeric substance matrix,on surface of food processing lines and instruments,enabling them to attach and survive on contact surfaces when food-processing environments treated with sanitization techniques.The biofilm can transfer contamination to food products and impose risk hazard to public health[105,106];L.monocytogenesstrains are capable of adhering to many materials found in food-processing plants,allowing their long-term persistence in food-processing plants[107];Moreover,the persistent strains are capable of surviving within food processing environment in high-risk production areas for longer periods of time(even years)[108];(D)Substantial proportions ofL.monocytogenesisolates derived from different foods and environmental sources are capable of being resistant to one or more commonly used antibiotics.They possess the capabilities of multidrug-resistance[56,109,120],imposing an additional risk and difficulty to public health.

Owning to these capabilities,variousL.monocytogenesstrains are capable of widely surviving and multiplying in the both normal and stressful environments in species- and subtypespecific manner[121].Their ubiquitous presence in theses environment settings can easily result in contamination of a wide variety of foods and environments and ultimately,the processed food products[47].In fact,variousL.monocytogenesstrains have been detected and isolated from a broad diversity of environmental sources,including soil,sewage/sewage sludge/river water[122,123],effluents[124],food-processing plants[125],decaying plants[126],and house environment[127].Contaminations of milk/dairy products[26,128,129],soft cheese and butter[37,130,131],RTE-meats/meat products[12,31,62,129,132],aquatic food products[27,129,133,134],various fresh/RTE vegetables[129,135–138],contaminated humans[139],animals[72],poultry[70]and human/animal feces[139,140]have been reported.Domesticated ruminants fed with silage contaminated withL.monocytogenesalso play a major role in maintainingListeria spp.in rural environment via a continuous fecal oral enrichment cycle[73,100].The dispersal of contaminated products of sewage treatment to agricultural fields and waters represents another important route for the transmission from their habitats to human populations[100].Food-processing environments can be also responsible for widespread of contaminated food sources available to consumers[140].For instance,Chapin at al.[11]detected 33% and 34%Listeriaspecies among of 588 and 734 samples collected from natural environments and food-production plants in New York State and found co-existence ofL.monocytogeneswith one or more of otherListeriaspecies in a given sample in 3% and 9% of samples from natural environments and foodproduction plants and a close association between isolation ofListeriaspp. and soil moisture/proximity to water and pastures in food production environments. Sauders et al.[121]identified 80L.monocytogenesand 50L.innocuaisolates among 442Listeriaisolates from 1805 soil,water,and other environmental samples collected from 4 urban and 4 rural areas representing natural environments over two years in the state New York,USA.Wu et al.[56]collected,analyzed and characterized 1036 samples,including raw meats,aquatic products,quickly frozen products,edible mushrooms,vegetables,collected from provinces in North and South China.They found that 20.9%(41/196)of meat/meat products;6.4%(18/280)of aquatic products,44.9%(88/196)of quickly-frozen foods;23.2%(52/224)of edible mushrooms and 5.7%(8/140)of vegetables were contaminated withL.monocytogenes.BecauseL.monocytogenesstrains are persistent in food processing plants,food-associated environments,farm environment,retail environment,private and public kitchens and general environment,they can be introduced into RTE foods via cross-contamination[141].Thus,the capabilities ofL.monocytogenesto multiply in diverse habitats,to survive under adverse conditions for long period of time,to spread and persist in broad environment and food sources,the general severity of listeriosis and the extreme difficulty in eradicating it plus the“zero-tolerance”policy forL.monocytogenesin RTE products in USA underscore the paramours importance of availability of specific,efficient,accurate and reliable methods for rapid detection and food-safety measures for control and prevention of this fatal foodborne pathogen in foods,food processing plants and environment settings[21,142].

3.Culture-based methodologies for isolation and identification of L.monocytogenes strains in foods and environment sources

Association of Official Analytical Chemists International Official Standard(AOAC-IOS)Methods Manual protocols have been used to screenListeria sp.orL.monocytogenesin foods.Some methods and references forListeria spp.andL.monocytogenesscreening are listed in Table 2A and B [143–161],respectively.Screening and isolation ofL.monocytogenescan also be done by Bacteriological Analytical Manual(BAM)Chapter10“Detection and Enumeration ofListeriamonocytogenesin Foods”(http://www.fda.gov/Food/FoodScienceResearch/Laboratory Methods/ucm071400.htm),developed by U.S.Food and Drug Administration(U.S.FDA);“Isolation and Identification ofListeriamonocytogenesfrom Red Meat,Poultry and Egg Products,and Environmental Samples”developed by U.S. Department of Agriculture(USDA)(http://www.fsis.usda.gov/wps/portal/fsis/topics/science/laboratories-and-procedures/guidebooks-and-methods/microbiology-laboratory-guidebook/microbiology-laboratory-guidebook);Methods included in the Laboratory Procedures for the Microbiological Analysis of Foods by Health Canada(http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/volume3-eng.php); Microbiology of the food chain Horizontal method for the detection and enumeration of Listeria monocytogenes and ofListeriaspp.by International Organization for Standardization (ISO,http://www.iso.org/iso/isocatalogue/cataloguetc/cataloguedetail.htm?csnumber=60313,ISO/DIS11290-1 and ISO/DIS 11290-2);Detection and enumeration of Listeria monocytogenes and other Listeria species by Public Health England(https://www.gov.uk/government/publications/detection-andenumeration-of-listeria-monocytogenes-and-other-listeriaspecies).

Table 2(A)Rapid screening methods for Listeria sp.(B)Rapid screening methods for Listeria monocytogenes.

In the BAM method,food or environmental samples are enriched at 30℃ for total 48h,and at 4h,selective agents are added.The selective agents contain nalidixic acid for inhibition of gram-negative bacteria,acriflavine for selectivity with grampositive bacteria and cyclohexamide for inhibition of yeast and mold(or pimaricin, an antifungal agent).Screening and isolation ofL.monocytogenesis performed from the enriched samples.Screening can be done by rapid methods such as VIDAS(bioMerieux),or by two types of selective media streaked after 24 and 48h,including one of esculin-based and chromogenic selective agar.Esculin-based agars are used to detectListeriaspp.as allListeria spp.can hydrolyze esculin to produce esculetin,a dark brown or black complex with ferric citrate.Oxford(OXF)and Palcam(PAL)Agars are the standard agars based on the ability of allListeriaspecies to hydrolyze esculin and their similar colony morphology on these agars.Thus,they can used to screen for allListeriaspecies.However,this method makes differentiation of pathogenicL.monocytogenesfrom other non-pathogenic species difficult.Table 3A lists the esculin-based agar[162–165].A number of selective agars have been approved for isolation ofL.monocytogenes,as shown in Table 3B[166–169].

Table 3(A)Esculin-based agar in the BAM.(B)Chromogenic agars in the BAM for Listeria monocytogenes.

Listeriaagar according to Ottaviani and Agosti(AL or ALOA medium,Bio-Rad)and BBLTMCHROMagarTMListeria,BD are selective media used for isolation,differentiation and identification ofL.monocytogenesandL.ivanoviistrains from foods and environmental samples.This method is based on:(a)selective inhibition of growth of gram-negative organisms,yeast and fungi by adding selective agents to culture media;and(b)simultaneous detection of activities of both β-glucosidase and phosphatidylinositol-specific phospholipase C(PI-PLC),a potential virulence factor present specifically in bothL.monocytogenesandL.ivanoviibut not in other listeria species.AL medium contains X-glucoside,a chromogenic substrate producing a blue-green colored compound when being hydrolyzed by β-glucosidase common and specific toListeriaspecies.X-glucoside hydrolysis by β-glucosidase results in formation of a blue to blue-green color in allListeriacolonies.PI-PLC is only detected inL.monocytogenesandL.ivanovii.AL medium or BBL CHROMagarListeriaplate contains phosphatidylinositol,a phospholipid substrate,which,when being broken down,produces an opaque halo around the colonies of these two species.The halo is visible after 24h forL.monocytogenesand 48h forL.ivanovii.One advantage of BBL CHROMagarListeriais that it is able to distinguishL.monocytogenesandL.ivanoviifrom otherListeriaspecies,thus,facilitating detection ofL.monocytogenesandL.ivanoviiin the presence of otherListeriaspecies and other bacterial flora in a sample.The risk of not detectingL.monocytogenesorL.ivanoviistrains can be minimized.The procedures and the results of CHROMagarListeriain detection ofL.monocytogenesandL.innocuastrains are shown in(Fig.1A).

Fig.1.AL(Agar Listeria according to Ottaviani and Agosti medium)Agar*.

RAPID’L Mono Agar(RLM,Bio-Rad Laboratories)is another chromogenic medium able to differentiateL.monocytogenesfrom otherListeriaspecies by a color change reaction.This medium can also be used for confirmation and/or enumeration ofL.monocytogenes.With this protocol,full results can be obtained in 48h using one plate and one broth and is suitable for the speciesL.ivanoviiinfrequently found in food matrices.Furthermore,selective solution in medium inhibits most of interfering flora,including Gram-positive and Gramnegative bacteria,yeasts and molds.The unique chromogenic principle due to PI-PLC activity and the xylose fermentation of this medium make enumeration and differentiation ofL.monocytogeneseasier.As shown in Fig.1B,blue colonies areL.monocytogenes(PIPLC+/xylose–)while blue colonies with a yellow halo areL.ivanovii(PIPLC+/xylose+)and white colonies with or without a yellow halo are otherListeria spp.(i.e.PIPLC–/xylose+or?).This medium is specific as other bacteria and yeasts are inhibited,allowing rapid and specific identification ofL.monocytogenesin 24h and of otherListeriaspecies in 24 and 48h,after preparing samples in compliance with standards detection and enumeration ofL.monocytogenesin 24 h after enrichment. This method has an excellent agreement with U.S.Department of Agriculture/Food Safety and Inspection Service;U.S.FDA/BAM;and AOAC[170].

If positive,five colonies from each plate are streaked on TSAYE plates for purity before further analysis.Confirmation can be performed after pure isolates are obtained using different methods,including wet mount,gram staining,CAMP test,catalase test,APIListeria(bioMerieux),VITEK,hemolysis analysis,serology test qPCR and sequencing.L.monocytogenesare divided serologically into 15 serotypes. As mentioned above,a majority of strains that cause listeriosis belong to the serotypes 1/2a,1/2b and 4b[171].Thus,subtyping ofL.monocytogenesmay play a critical role in providing epidemiological information for outbreak investigation[94,172,173].

More recently,Yang et al.[174]described a multivalent vancomycin-conjugated brush-like magnetic nanoplatform which can offer highly efficient enrichment with good purity forL.monocytogenes.This enrichment platform coupled with PCR-ECL detection enables rapid and accurate detection ofL.monocytogenesin complex samples with a LOD of 10 cfu/mL.

Currently,the traditional culture enrichment is still the most commonly used approach for detection and isolation ofL.monocytogenesfrom food matrices prior to detection with PCR-based or other methods described below. For BAM culture enrichment,the selective media containing acriflavin(10mg/L),cycloheximide(40mg/L) and sodium nalidixic acid(50mg/L)are utilized to promoteL.monocytogenesgrowth while simultaneously suppressing the growth of other cohabitating non-target bacteria in food matrices being tested.However,inhibitory components of food matrices still remain during the entire enrichment period even though they are diluted,which can influence the capability ofL.monocytogenesto multiply to detectable levels,causing possible false-negative results.The other disadvantage is that when selective agents are added to culture media,they do not completely inhibit the growth of all the microbes naturally present in food metrics.Because the number ofL.monocytogenescells present in food metrics is generally very low[175],it is likely that other food microbiota,including the five nonpathogenic species ofListeria,can impose negative impact on growth ofL.monocytogenesvia competition for nutrients.Indeed,L.monocytogenesgrowth was reported to be strongly inhibited due to the presence of otherListeriaspecies,e.g.L.innocuaand/orL.welshimeri[176–179]and other competing microorganisms occurring naturally in foods[180].Since rapid and sensitive detection ofL.monocytogenesis of paramour importance in the food industry and medical diagnosis,traditional culture enrichment methods do not completely meet such urgent requirement,new approaches are needed.

4.Other methodologies for detection and characterization of L.monocytogenes

4.1.Detection of L.monocytogenes with antibody based-assay and aptamer-based assays

AsL.monocytogenesstrains contain specific surface proteins,including somatic(O)-and flagellar(H)-antigens,it is likely to detect them by using individual and/or combinations of antibodies specifically against these antigens[94].

Standard sandwich immunoassay designed for detection ofL.monocytogenesgenerally involves formation of an immunecomplex between an immobilized primary antibody,targetL.monocytogenesand a labeled secondary antibody.The primary antibody is firstly immobilized on beads,e.g.gold particles,magnetic beads,silica,polystyrene microtiter plate,filter membranes,or directly on surface of transducers.Enzyme-linked immunosorbent assays(ELISAs)are generally conducted in 96-well polystyrene plates to which the primary antibodies or proteins bind.The binding and immobilization of ELISA reactants to microplate surface allows easy separation of the bound antigen from non-bound materials.This ability to wash away nonspecifically bound materials makes the ELISA a powerful tool for detecting specific antigen(s)present in a crude preparation.A detection enzyme,e.g.horseradish peroxidase(HRP),alkaline phosphatase(AP),acetylcholinesterase,βgalactosidase,and catalase or other tags e.g.streptavidin,can be linked directly to the primary antibody or introduced via a secondary antibody recognizing the primary antibody or if the primary antibody is labeled with biotin for streptavidin.L monocytogenesstrains are detected by assessing the activity of conjugated enzyme via reaction with a substrate to produce a measureable product.The key element of this assay is a highly specific antibody-antigen interaction.A number of substrates with an HRP or AP conjugate are available for performing ELISAs.

Several ELISAs have been applied in detection ofL.monocytogenes[151,153,154,181–183].For instance,Curiale et al.[151]reported an early collaborative evaluation of Listeria-Tek ELISA for detection ofL.monocytogenesandotherListeria spp.in food samples.Later,VIDAS?Enzyme-Linked Immunofluorescent Assay(VIDAS LIS)was validated by a collaborative study involved in 19 laboratories for detection ofListeriain foods[154].VIDAS?LIS was compared with U.S.FDA culture method for detection ofL.monocytogenesin cheese,green beans,ice cream,and fish,ground turkey and roast beef,which were naturally contaminated or artificially inoculated withL.monocytogenesstrain at three concentrations.Of 1558 samples tested,935 were positive,including 829 by VIDAS and 809 by standard culture method.The overall negative rates for VIDAS?LIS and culture method were 10.3 and 13.5%,respectively,and 86% agreement between both methods.This study revealed that the sensitivity of VIDAS?ELISA was significantly higher than that of AOAC 993.12 in detection ofListeriain ice cream and that of USDA/FSIS method in detection ofListeriaat low contamination level in raw turkey.The positive results are regarded as presumptive and need to be further confirmed by isolation and identification ofListeriafrom the enrichment broth.“AOAC Official Method 999.06Listeriain Food”performed in the automated VIDAS?Instrument has been adopted and used for screening and detection ofL.monocytogenesin dairy,seafood,meat products,seafood and other foods.An enzymelinked fluorescent assay named VIDAS?LMO2 assay specific forL.monocytogeneshas been commercially available.Ueda and Kuwabara[184]applied this assay to detectL.monocytogenesin food samples and reported that this assay detected 105CFU/mL in less acidic food samples and 106CFU/mL in acidic foods samples(e.g.yogurts and fruit juices)artificially inoculated withL.monocytogenesand enriched for 12h.This assay,which can be finished in 70min,has been included in US FDA BAM for Detection and Enumeration ofL.monocytogenes[185].

Several studies[153,181,182]on food products and typical environmental surfaces found in a food-production facility by the AssuranceListeriapolyclonal enzyme immunoassay(EIA)and by BAM or USDA culture method for detectingL.monocytogenesand relatedListeriaspecies representing a variety of food products by Visual Immunoprecipitate Assay(VIP).The results indicate that the AssuranceListeriaEIA method and the USDA/FSIS culture method are equivalent for detection ofL.monocytogenesand relatedListeriaspecies from environmental surfaces.L.monocytogenes-specific antibodies linked to paramagnetic polystyrene beads[186]or nanoparticles[187,188]were applied in immunomagnetic separation(IMS)ofL.monocytogenescoupled with qPCR in detection ofL.monocytogenes.Lin et al.[189]generated a monoclonal antibody (mAb) specifically recognizing a 77-kDa surface protein ofL.monocytogenes.An immunoassay with this mAb showed great potential for identification,detection and serotypingL.monocytogenesserotype 4b.Tu et al.[190]recently reported isolation and expression of two novelL.monocytogenes-specific clones(L5-78 and L5-79)with high thermal stability,pH tolerance,and urea resistance from a phage display antibody library.Both clones recognized 1/2a,1/2b,and 4b serotypes ofL.monocytogenes.An ELISA with clone L5-79 could detectL monocytogenesin pasteurized milk with a LOD of 1×104CFU/mL.

One advantage of immunoassays is that they can be performed in multiplex formats,and thus,reduce analysis time,reagents and costs for detection of multiple foodborne pathogens.For instance,Magliulo et al.[183]applied a sandwich ELISA to simultaneously detectL.monocytogenes,Salmonella enterica,E.coliO157:H7,andYersinia enterocoliticawith four monoclonal antibodies specific for these bacteria simultaneously immobilized in a 96-well plate.Its LODs for these bacteria ranged from 104to 105CFU/mL.However,several disadvantages are associated with immunoassays.One of them is that they are less sensitive,as their sensitivities are generally in the range between 105and 106CFU/mL[172].Other drawbacks are related to cross-reactivity of antibodies due to possible antigen-sharing between closely related strains,the high cost of generating specific antibodies and variations in expression levels of surface H-and O-antigens[172].

Aptamers can be a promising alternative to antibodybased immunoassay.Aptamers are single-stranded DNA or RNA oligonucleotides binding to a variety of targets ranging from single small molecules to whole cells with high affinities and specificities[191].Aptamers are selected from a library by anin vitroprocedure called systematic evolution of ligands by exponential enrichment(SELEX)[192].Aptamers possess advantages over protein-or antibody-based assays,including high affinity and specificity for targets,good stability,ease of chemical synthesis and modifications,easy storage/transportation[193].For instance,Ohk et al.[194]selected and applied aptamer-A8 specific for internalin A(Table 4),an invasive protein present on the surface ofL.monocytogenes,in the fiber-optic sensor coupled with antibody in a sandwich format to detectL.monocytogenesin foods.P66,a biotinylated polyclonal anti-Listeriaantibody,was immobilized on optical waveguide surface coated with streptavidin for capturingL.monocytogenes,and A8 conjugated to Alexa Fluor 647 was used as a reporter.The biosensor selectively detected pathogenicListeriain pure culture,a mixture with other bacteria with LOD of 103CFU/mL and alsoL.monocytogenescells initially inoculated with 102CFU/25g RTE turkey and sliced beef and chicken meats after 18h enrichment.Ding et al.[195]developed and applied an label-free electrochemical potentiometric aptasensor to detectL.monocytogeneswith an aptamer binding specifically to internalin A(Table 4).The target-binding event prevented aptamer from electrostatically interacting with protamine,which was sensitively detected using a polycationsensitive membrane electrode.Using this method,10 CFU/mL ofL.monocytogeneswere detected.In combination with an online filtration system,bioassay displayed good recovery and high accuracy after being evaluated with spiked coastal seawater samples.With whole-bacterial SELEX,Lee et al.[196]selected two aptamers(Table 4)with high affinity and specificity for liveL.monocytogenes(Table 4),which displayed an enhanced sensitivity and good linearity within the range of 20–2×106CFU/mL.Zhang et al.[197]developed a aptamer-based method based on Fe3O4nanoparticle cluster(Fe3O4NPC)-catalyzed signal amplification.With this method,L.monocytogeneswas first captured with vancomycin(Van),a glycopeptide antibiotic for Gram-positive bacteria as the first molecular recognition agent.L.monocytogenes-specific aptamer(Table 4)modified by Fe3O4NPC was used as the signal amplification nano-probe.BecauseL.monocytogenescan be recognized by both Van and aptamer at different sites,the sandwich recognition showed high specificity.Fe3O4-NPC displayed collective effect-enhanced catalytic activity for the color reaction of chromogenic substrate.The change in absorbance or color is indicative forL.monocytogenesconcentration.With this method,L.monocytogeneswhole cells could be directly assayed with good linearity within the range of 5.4×103–108cfu/mL with a visual LOD being 5.4×103CFU/mL.

Table 4Sequences of Aptamers for L.monocytogenes.

4.2.LOOP-mediated isothermal amplification(LAMP)for detection of L.monocytogenes and L.ivanovii

Unlike conventional and qPCR-based technologies in which PCR is carried out with a series of alternating temperature steps,LAMP is an isothermal DNA-amplification technique with high specificity,efficiency and rapidity at constant temperature(isothermal)(60–65℃).LAMP employs two inner primers and two outer primers to identify 6 distinct regions on target gene with Bst DNA polymerase,which possesses a large fragment with a high strand displacement activity and a replication activity.An additional pair of“l(fā)oop primers”can be used to further accelerate the reaction[198,199].Because of the specific nature of their actions, the amount of DNA amplified in LAMP is considerably higher than that of PCR-amplified products.During LAMP,an increasing amount of magnesium pyrophosphate precipitate is generated as a byproduct in solution,represented as turbidity,which allows easy visualization by naked eye for larger reaction volumes or can be detected via photometry for smaller volumes[200]or can also be monitored in real-time manner by either measuring turbidity[201]or fluorescence using intercalating dyes such as SYTO green to generate a change in visible color,which can be seen with naked eyes or can be more accurately measured by instrumentation[202].Dye molecules intercalate or directly label the amplified DNA,which,in turn,can be correlated proportionally to DNA copy numbers initially present.

A number of studies[203–209]have applied LAMP to detectL.monocytogenes.For instance,Wang et al.[204]developed and applied ahlyAgene-based LAMP assay(Table 5)to detectL.monocytogenesin pure culture and chicken specimens artificially contaminated withL.monocytogenesstrains.LAMP assay highly and specifically detected fourL.monocytogenesstrains but did not detect 16Listeria spp.and 13 non-Listeriastrains,and was significantly faster than was conventional PCR assay,as LAMP took 90min for the entire procedure while PCR assay took 160min.When being applied to identifyL.monocytogenesthrough white precipitate of magnesium pyrophosphate in reaction tubes,LAMP showed 100 folds more sensitive than did conventional PCR.Compared with conventional PCR,LAMP assay was more rapid and highly sensitive in detectingL.monocytogenes.Tang et al.[203]applied LAMP to detectL.monocytogenesin microbiology laboratory and the field.They found that LAMP was 100 times more sensitive than PCR method as it detected 2 CFU/reaction.Zhang et al.[205]developed ahlygene-based LAMP(Table 5)and applied it to detect 88L.monocytogenesisolates,1 reference strain and 33 non-targeted bacterial strains via comparing it with qPCR and conventional ISO11290-1 method.The detection results of LAMP and qPCR for 89L.monocytogeneswere 100% positive and for 33 non-targets bacteria strains were 100% negative with the sensitivity of LAMP being 200 CFU/mL,comparable to qPCR method but better than conventional method.Wang et al.[208]designeda prfAgene-based LAMP(Table 5),and used dimethyl sulfoxide and touchdown LAMP to increase its sensitivity and specificity.Its LOD was 10 fg/reaction,a tenfold higher than a commercial Isothermal Amplification Kit and a 100-fold more sensitive than previously reported LAMP assays.This assay was able to detect 11L.monocytogenesstrains with negative reaction to otherListeriaspecies,includingL.innocua and L.invanovii.Wu et al.[206]developed a double LAMP(dLAMP)based on bothhlyAandiapgenes(Table 5).Its LOD forL.monocytogenesat 63℃ for 15min was 10 fg DNA/reaction.With normal LAMP ofhlyAoriap,the LOD was 100 fg DNA/reaction.Furthermore,mineral oil and Gold View II nucleic acid stain were used as basic materials to develop a simple visualization of positive samples.When applied to test 450 food samples forL.monocytogenes, dLAMP protocol displayed accuracy identical to that of culture-based method.A multiple inner primer-LAMP system with 10 specific primers targeting 14 different regions(Table 5)was recently developed and applied to detectL.monocytogenes[209].This system was capable of efficiently amplifying the target elements in genomic DNA fromL.monocytogenesat 63℃ for 35min with LOD of 62.5 fg with purified DNA,2.4 CFU/reaction in buffer and 24 CFU/reaction in milk,respectively.When being applied in testing 48 pork samples,its diagnostic accuracy was 100% as compared to the culture-biotechnical method. Miks-Krajnik et al.[210]validated a commercial LAMP coupled with bioluminescence for the detection ofL.monocytogenesat low levels on food contact surfaces.They found that this method performed as effectively as ISO method,exhibiting 100% specificity and sensitivity with LOD of 10 CFU/100cm2.

Wang et al.[207]developed and evaluated LAMP for detection ofL.ivanoviistrains using three primer sets targetingsmcLgene ofL.ivanoviispecies(Table 5).LAMP-amplified prod-ucts were directly viewed by LOOPAMP fluorescent detection reagent or detected by agarose gel electrophoresis.The turbidity of LAMP reactions was monitored in a real-time manner.LAMP was completed in 1h at 64℃ with inclusivity of 17L.ivanoviistrains and exclusivity of 77 non-L.ivanoviistrains being 100% and LODs being 250 fg DNA/reaction in pure cultures or 16 CFU/reaction simulated human stool,which was 100 and 10 folds higher than those of conventional and qPCR assays,respectively.When being applied to human stool samples spiked with 16 CFU/g ofL.ivanovii,this LAMP achieved positive detection after 6h enrichment,indicating its usefulness in rapid detection ofL.ivanovii.

Table 5(A)List of sequences of oligonucleotide primers for LAMP.(B)Primers used for MIP-LAMP amplification.(B)Primers used for MIP-LAMP amplification.

An advantage of LAMP is that its amplification efficiency is high because of its no-time loss for thermal change due to isothermal reaction.Thus,it displays specificity,selectivity and rapidity higher than those of other DNA-based amplification methods.LAMP can be accelerated by introducing forward loop primers[199,200].However,its applications may be limited by the presence of inhibitors in raw samples and its lower sensitivity as compared with PCR-based methods.

4.3.Detection of L.monocytogenes with matrix-assisted laser desorption ionization-time of flight-mass spectrometry(MULDI-TOF-MS)

Application of MALDI-TOF-MS in detection ofL.monocytogenesis based on generation of profile spectra of small proteins/peptides mainly derived from bacterial ribosomes.Initiating from crude bacterial extracts,cell lysates,or even whole cells,MALDI-TOF MS spectra can be acquired to reveal the characteristic peaks for each bacterial species and, in some cases,even for subspecies or clonal complexes[211].This method has been applied in rapid identification of foodborne microorganisms,by comparing a signature of biomolecules against database[212–215].

The VITEK MS?(bioMerieux,Durham,N.C.)is MALDITOF-MS that can rapidly identify microorganisms from cultures.Proteomic analysis using this MALDI-TOF-MS can be performed directly from colonies once they are isolated,therefore,reducing the turnaround time for microbial identification.The characteristic peaks of each organism resulted from mass spectrometry are used for identification by comparing them against the FDA-approved database using BIN system.Generally,the system uses mass spectra of the mass rangem/zfrom 2 to 20kDa representing ribosomal proteins[211].The microorganisms are identified without needing to obtain specific peaks.When compared with conventional phenotypic methods,MALDI-TOF-MS provides an equal or higher accuracy[216,217].Isolated colonies of presumptiveL.monocytogenesgrown on a TSAYE agar are selected and re-streaked on a 5% sheep-blood plate agar for 24h.The colonies are then applied on a spot on a target slide,followed by collection of mass spectrometry peaks with the VITEK MS?.The identification is then performed by the MYLATMsystem within a few minutes.The results are shown in Fig.2.This method has been proved to be liable and the fastest one for microbial identification.MALDI-TOF-MS has been used in identification ofL.monocytogenesin milk samples spiked with single species and multiple species cultures and enriched in a selective enrichment broth for 30h.A few CFU ofL.monocytogenes/mL of initial selective broth culture could be detected.When the same approach was applied to solid foods previously implicated in listeriosis,the detection was achieved in the same time interval at inoculation levels of 10 CFU/mL[218].MALDI-TOF-MS can be used in source-tracking ofL.monocytogenes[218].However,its wider applications are limited by lower power in differentiation of closely related isolates,the dynamic variations in protein expression levels,and a lack of appropriate mass spectra in database[219].

Fig.2.The VITEL?MS report and mass spectrogram for L.monocytogenes identification.

4.4.Detection of L.monocytogenes with microarray and whole genome sequencing(WGS)via next generation of DNA sequencing

During the last decade,microarray technology has been applied in genomics study.Using Affymetrix platform,the USFDA has played a significant role in custom designing theListeriaGeneChip to explore the genetic diversity ofL.monocytogenes.Investigation of genomic diversity of many prokaryotic microorganisms using Pan-genomics has been reported[220].Microarray technology has been applied in study of gene presence and absence ofL.monocytogenes,resulting in molecular serotyping[221].Over 80L.monocytogenesstrains in FDA collection from the main pathogenic serotypes,including1/2a,1/2b and 4b,have been analyzed usingListeriaGeneChip to identify their relatedness.The results revealed that these strains are separated into their respective serotypes.When using neighbor net method to analyze 31 strains from serotypes 1/2b and 4b as they are more closely related than the 1/2a strains,the serotype 4b strains are also divided into four distinct clusters corresponding to their ECs[221].

WGS,one of important methods toward understanding ofL.monocytogenes,is beneficial to assistL.monocytogenesoutbreak investigation in that WGS analysis can be used to compare the environmental and clinical isolates[222].With advent of second generation sequencing techniques, the numbers of microbial genome sequences have begun to accumulate in public data bases[223,224].The advancement of technology used for DNA sequencing via next generation sequencing has made it possible to sequence the entire genome of aL.monocytogenesstrain within a short time and at a reasonable cost[223].The results ultimately lead to development of multiple techniques with varying degrees of data integration with distinct possibility for automation and a reduced cost.The process was successful with the increased computational power and the ailability of sophisticated bioinformatics tools.A large number of WGS ofL.monocytogeneshave been available in various public database representing several outbreaks and non-outbreak associated strains[225].

4.5.Detection of L.monocytogenes with genetically engineered bacteriophages

Bacteriophages are viruses that are natural enemies of corresponding host bacteria.They specifically infect host bacteria and usually cause the disintegration or dissolution of bacterial hosts.Based on these unique and specific properties,bacteriophages can be ideally suited for development of highly specific assays for detection ofL.monocytogenes.To date,several genetically engineered phage systems have been developed and applied in detection ofL.monocytogenesstrains[226,227].For instance,Loessen et al.[228])constructed a recombinant derivative of bacteriophage A511,agenus-specific,virulent myovirus infecting 95% ofL.monocytogenesserovar 1/2 and 4 cells,by inserting luxAB carrying the gene for a fusedVibrio harveyiLuxAB protein immediately downstream of the major capsid protein gene.L.monocytogenescells were infected with this A511 derivative for 2h,5×102to 103cells/mL were detected by using a single-tube luminometer.Moreover,after an enrichment step,＜1 cell/g could be detected.Stambach et al.[229]developed aListeriadetection method by utilizing A511 bacteriophage amplification coupled with surface-enhanced Raman spectroscopy(SERS)and lateral flow immunochromatography.In this assay,antibodies specific for A511 were covalently linked to SERS nanoparticles.The resulting antibody-conjugated SERS nanoparticles were printed onto nitrocellulose membranes and used as quantifiable reporters.When A511 was present,phage-SERS nanoparticle complexes were arrested and concentrated as a visible test line,which could be interrogated quantitatively by Raman spectroscopy.An increase in captured phage-reporter complexes was correlated by corresponding increase in SERS intensity with LOD of 6×106pfu/mL.Cappillino et al.[226,230]successfully engineered the bacteriophages specific for the six species of genusListeriaby inserting mutated luciferase gene into the genome of bacterial phages.When engineered bacteriophages are incubated withListeriastrains,they specifically bind and internalize intoListeriacells within which they rapidly replicate their genomic DNA and synthesize their own DNA-encoding proteins,including the engineered gene for luciferase,which emits blue light while catalyzing the reaction with its substrate.Based on this specific bacteria-bacteriophage parasitic-host partnership,the presence and the quantity ofListeriaspecies in food and environmental samples can be readily and rapidly detected by measuring the emitted light with Luminometer.The luciferase activities expressed by engineered bacteriophages are proportional to the amounts of light emitted,which,in turn,reflects the number ofListeriacells.An assay kit based on these engineered bacteriophages cocktail named Sample6 DETECT/L has been commercially available.

5.Methodologies for subtyping L.monocytogenes strains

The virulence potential and ecology ofL.monocytogenesisolates can be predicted to a large extent by using different subtyping methods,including serotyping,pulse field gel electrophoresis(PFGE),multi-locus sequence typing(MLSC),ribotyping,phagetyping,WGS and 16S rRNA gene sequencing etc.

L.monocytogenesstrains are generally serotyped based on the variations in their O-and H-antigens and assigned to their lineages using of genotypic and phenotypic approaches,and can be classified into four lineages:lineage I(serotypes 1/2b,3b,4b and 3c);lineage II(serotypes 1/2a,1/2c,and 3a);lineage III(serotypes 4a,4b;and 4c)and lineage IV(4a,4b,and 4c),respectively[3].Strains with serotypes 1/2a,1/2b,and 4b have been most frequently isolated from food-related sources while serotype 4b isolates have been known responsible for most human listeriosis epidemics and human sporadic cases[5,25,231,232].Thus,identification of strain serotype will enable differentiation among important foodborne strains(e.g.1/2a,1/2b,and 4b)and provides a“gold standard”for comparing isolates analyzed by different laboratories and with different methodologies[233].Typing techniques are assessed based on their performances and convenience of use in term of discriminatory power,reproducibility,typeability,and agreement between typing techniques,cost,availability of reagents/equipment,rapidity,ease of execution interpretation of results and versatility etc.

5.1.Serotyping

Serotyping with related antibodies is an accepted subtyping method useful for subtypingL.monocytogenesand for addressing epidemiological considerations.MostL.monocytogenesisolates collected from the environment sources and patients are types 1 and/or 4.More than 90% ofL.monocytogenesisolates can be serotyped with commercially available antibodies being used to characterize isolates such as serotypes 1 and 4 or serotypes 3,5,and 6 etc.However,the disadvantage of serotyping with antisera include(i)possible cross-reaction,becauseL.monocytogenesshares one or more somatic antigens with all the nonpathogenicListeriaspecies,and(ii)limited discriminatory power and lower accuracy,which are largely dependent on specificity and quality of the antisera[172].

5.2.Genetic subtyping

5.2.1.Subtyping and characterization of L.monocytogenes strains with pulsed- field gel electrophoresis(PFGE)

Among the available subtyping techniques,PFGE is regarded as“gold-standard”method for subtyping ofL.monocytogenesfor tracking source and epidemiologic investigation because of its excellent discrimination power and reproducibility[234–236].PFGE is an agarose gel electrophoresis technique for separating larger DNA fragments by applying electrical current that periodically changes three directions in a gel matrix.The standardized PulseNet PFGE procedures(http://www.cdc.gov/pulsenet/PDF/listeria-pfge-protocol-508c.pdf)forL.monocytogeneshas been described[236],which include:(a)preparation of genomic DNA;(b)digestion of genomic DNA in agarose plugs slices with restriction enzymes Asc I or Apa I or SmaI;(c)separation of restriction DNA fragments in plugs by electrophoresis in agarose gel;(d)staining and de-staining of agarose gel;and(e)analysis of the PFGE patterns using the BioNumerics software.Tag image file format(TIFF)images are normalized by aligning the peaks of size standard strain.For instance,a diverse population ofL.monocytogenesisolates was identified by using multiple PFGE in the 44 isolates from retail meat in Detroit,US[118].PFGE has been applied in subtyping ofL.monocytogenesisolates from a variety of foods,e.g.retail raw RTE foods,infant foods,raw chicken in China[56,120]and raw foods in Chile[237].Using PFGE,Nucera et al.[238]subtyped 300L.monocytogenesisolates collected from foods in Italy from 2003 to 2007 and generated 164 combined PFGE profiles.Serotypes 1/2a,1/2c and 4b/4e were the most frequent ones,accounting for 45,22,and 16%,respectively while serotype 1/2a was significantly associated with dairy samples and 1/2c was significantly associated with meat samples.By applying PFGE,Miettinen et al.[239]distinguished two different PFGE types forL.monocytogenesfrom an ice cream plant according to the information obtained with three restriction enzymes and found that the dominant PFGE type was persistent in ice cream plant for seven years.

While PFGE typing could provide the most sensitive strain discrimination and has become standard subtyping method for detection of listeriosis outbreaks,it is time-consuming,laborintensive and tedious in the interpretation of PFGE patterns,and requires special equipment.Thus,PFGE is practically performed after serotyping.

5.2.2.Multi-locus sequence typing(MLST)and phylogenetic analysis

MLSTs based on nucleotide sequences of either housekeeping genes[240]or multi-virulent genes[241]are discriminatory forL.monocytogenesstrains.The protocol of MLST involves:(a)isolation of genomic DNA from overnight cultures ofL monocytogenesin BHI broth using genomic DNA kits;(b)PCR amplification of nucleotide sequences of internal fragment of housekeeping genes(e.g.ABC transporter,superoxide dismutase,l-lactate dehydrogenase,amino acid aminotransferase,phosphoglucomutase,succinyl diaminopimelate dessucinylase,β-glucosidase,and histidine kinase and catalase based on DNA sequences of these loci from GenBank using the primers designed with software[240])or multivirulent genes[241]listed in Table 6 Zhang et al.[241]developed a MLST protocol based on internal fragments of three virulence genes(prfA,inlB,and inlC)and three virulence-associated genes(dal,lisR,and clpP)with the primer pairs targeting the known sequences ofL.monocytogenes strainEGD-e(Table 6)(http://www.pasteur.fr/recherche/genopole/PF8/mlst/Lmono.html).A 500-bp internal fragment of each gene was amplified to allow accurate sequencing of a 450-bp fragment within each gene using the procedures similar to those described above.A cluster diagram can be constructed.Doijad et al.[242]performed MVLS of 25L.monocytogenes4b isolates among 156L.monocytogenescollected from different sources in India over a 10-year period by using the primers,PCR conditions and procedures for MVLS described by Zhang et al.[241].They reported that 25 isolates could be classified into three clusters:Cluster I comprised 21 isolates including 9 animals,4 human,and 8 foods,which matched epidemic Clone I(ECI)while remaining three isolates formed another cluster.A single animal isolate was positioned into VT98 and VT99,respectively, suggesting that ECI isolated from different sources and places has persisted in India for＞10year.By applying this MVLS,Miyaet al.[58]conducted the genetic characterization of clinicalL.monocytogenesisolates and identified MVLST profiles of 7 clinical isolates identical to ECI isolates,which caused serious outbreaks in other countries.For instance,ECI isolates share their genotypes with Japanese isolates.

Table 6Primers used for multilocus sequence typing for L.monocytogenes.

MVLST can provide the higher discriminatory power and resolution to RT-PCR and/or PFGE-indistinguishable strains by at least one allelic difference and can provide unambiguous,inter-laboratory comparable results via internet.Its disadvantages is that the tools and algorithms are not available for analysis of MLST data and for accommodating variable amounts of recombination,which remains a hindrance limiting its wider applications in subtypingL.monocytogenes[243].

5.2.3.Ribotyping

Ribotyping ofL.monocytogeneswas initially developed by Bruce et al.[244]and Hubner et al.[245].Its procedures included:(a)L.monocytogeneswere classified using taxonomic characters derived from rRNA generated from digestion of genomic DNA ofL.monocytogeneswith EcoRI.The resulting fragments were separated by gel electrophoresis,immobilized on a membrane,and hybridized with a labeled rRNA operon fromE.coli;(b)The images of patterns,positions,and intensities of hybridized fragments were acquired electronically;and(c):the band positions relative to standards and scale of signal intensity were normalized with software and background was reduced so that each strain was reproducibly represented in a database as a pattern. With these methods,L.monocytogeneswas resolved into 50 pattern types differing in the length of at least one polymorphic fragment.Pattern types representing multiple strains were recorded as the mathematical average of the strain patterns and arranged by size polymorphisms of assigned rRNA regions into subsets,revealing the branching genetic structure of the species.Subtracting polymorphic variants of a specific assigned region from pattern types and averaging the types within each subset resulted in reduced sets of conserved fragments that could be used to recognize strains of the species.Pattern types and reduced sets of conserved fragments were conserved among different strains ofL.monocytogenesbut were not observed in total among strains of other species.Analysis of 1346 strains with ribotyping method revealed the patterns,fragment sizes,and their frequencies of occurrence in the patterns.Statistical analysis of fragment sizes led to generation of unobserved combinations of bands,and predicted pattern types.The observed fragment bands revealed positions of EcoRI sites relative to rRNA sequences.Each EcoRI site had an occurrence frequency,and unobserved fragment sizes were postulated based on knowing the restriction site locations.The result of the recursion process applied to the components of strain data was an extended classification with observed and predicted members.By applying this method,Wiedmann et al.[98]observed 23 different types of ribotyping patterns for 133 strains tested.Five of which had not been described for any of 1346L.monocytogenesstrains classified by ribotyping[244].Fifteen ribotypes were found among 64 ruminant isolates.All the ribotypes which had a frequency of 0.05 in survey of 1346L.monocytogenesstrains were also represented among these ruminant isolates 4[244].

While ribotyping is useful for source tracking and epidemiological investigation with reproducible results and higher discriminative power,it has several disadvantages,including requirement of two restriction enzymes for higher discrimination,inefficient differentiation between1/2b and 4b serotypes and inter-laboratory variations[94,172].

5.2.4.Phage-typing

Since the first discovery of bacteriophages specific forListeria,several phage typing systems forL.monocytogeneshave been described and applied in epidemiological studies related toL.monocytogenes[246–250].Jasinska et al.[251]isolated 3 bacteriophages fromL.monocytogenesserovar 5 and 8 bacteriophages fromL.innocuafrom lysogenic strains.Phagovar determinations with these phages and 12 other phages isolated fromL.monocytogenesserovars 1/2 and 4b revealed different phage patterns that allowed distinction betweenL.monocytogenesandL.innocua.L.monocytogenesserovar 5 was highly sensitive to phages from lysogenic strains of various serovars.Sword et al.[252]examined lytic spectra of 11 phages against 149 strains ofListeriaand used a classification system with five of these phages to place 127 of these strains in 8 phage types.They found that phage susceptibility could be closely related to the serological type of the strain but not related to the animal source or geographical origin,suggesting thatListeriaphages can be used as a way for generic identification and/or as an alternative to serological typing in epidemiological investigations.Loessner et al.[249]developed a bacteriophage typing protocol for differentiatingListeriaisolates collected from dairy products and various foods.They used sixteen phages isolated from both environmental sources and lysogenic strains for typing according to their lytic spectra and divided them into four groups according to their host ranges.The phages of group I had a very broad lytic spectrum as they lysed almost all typeable strains while Group II phages had much more restriction in their host range as they lysed mainlyL.monocytogenesstrains.The phages of group III were the most useful for discrimination,as their host range was narrower.All the temperate phages belonged to this group,whereas groups I and II were composed exclusively of environmental phages.All the phages in group III were capable of lysing members of eachListeriaspecies tested,with the exception ofL.seeligeri,of which none of 13 strains tested was lysed by phages B012,B035 or C707.Phage A500 has an extremely restricted host range.Few strains were attacked,and those which were attacked were eitherL.monocytogenesorL.innocua.

The conventional phage typing included(a)phage propagation;(b)titration and(c)typing[249,250],which were subsequently modified into “reversed phage typing procedure”and applied in typing over 1000 strains ofListeriaincluding an extended set of 21 genus-specific bacteriophages with the overall typability of strains was 89.5%[250].Estera et al.[253]evaluated phage typing of 225Listeriaisolates with both conventional and“reversed”phage typing procedures.They found that 199 out of 225 isolates shared the same bacteriophage patterns while 5 exhibited different phage reactions,and the remaining 21 isolates were untypeable,with the overall typeability rates being 90.7% and 97.6% of the typeable isolates that shared the same phage patterns by both procedures.Reversed phage typing had been applied in phage-typing ofL.monocytogenesin European red smear cheese[254]to differentiate strains,determine and compare their sensitivities to a bacteriophage mixture(LMP-102)containing six distinct lytic phages specific forL.monocytogenesserotypes 1/2a,1/2b,1/2c,3a,3b,4b,and 4d[132].Chen et al.[117]reported thatL.monocytogenes-specific bacteriophage cocktail were capable of lysing 65 of 91L.monocytogenesisolates derived from RTE foods.A panel with 26 phages termed “the international phages”for phage typing of serogroup 1/2 strains and 14 phages for the phage typing of serogroup 4 isolates has been recommended for the international method for phage typing[255].

However, several major disadvantages associated with phagetyping include:(a)a large sample set of phages is needed which is available only in reference laboratories;and(b)a fraction of strains ofL.monocytogenessubtypes are resistant to phage lysis and are untypeable, as it has been reported that persistentL.monocytogenessubtypes isolated from a smoked fish processing facility included both phage susceptible and resistant isolates[256].

5.2.5.Subtyping with whole genome sequencing(WGS)and 16S rRNA gene sequencing

WhileL.monocytogenescan be serotyped into major diseasecausing serogroups by a standard multiplex PCR technique[93],which,coupled with microarray analysis and slide agglutination,can be used in quickly identifying serotypes of majorL.monocytogenesserotypes 1/2a,1/2b,1/2c,3a,3b,3c and 4b[225,257],the recent emergence of WGS technologies has led to development and applications of new high-resolution typing schemes for comparison of bacterial isolates in outbreak detection,characterization and subtyping ofL.monocytogenesstrains,because WGSs of a number ofL monocytogenesisolates have been available[257–265].Ruppitsch et al.[262]defined and assessed a core genome multilocus sequence typing scheme for WGS-based typing ofL.monocytogenes.By comparing WGSs of 423L.monocytogenesisolates,Kwong et al.[263]found a diverse genetic population structures derived from three distinct lineages.Distinct nested clusters within groups of isolates which are indistinguishable with other typing methods could be identified by using WGS.When being applied in prospective epidemiologic surveillance on a panel 97L.monocytogenesisolates over a 12-month period,WGS provided a higher level of discrimination than that provided by conventional typing for inferring linkage to point source outbreaks.Furthermore,a risk alert system based on WGS similarity could be used to inform epidemiologists required to act on the data,suggesting the possibility of adopting WGS for prospectiveL.monocytogenessurveillance.Moura et al.[266]developed a universally applicable genome-wide strain genotyping approach and investigated the population diversity ofL.monocytogenesusing 1696 isolates from diverse sources and geographical locations. They defined the population structure ofL.monocytogenes,demonstrated the occurrence of international circulation of strains and revealed the extent of heterogeneity in virulence and stress resistance genomic features among clinical and food isolates.Using historical isolates,they showed that the evolutionary rate ofL monocytogenesfrom lineages I and II was low(about 2.5×10?7substitutions/site/year,as inferred from the core genome)and that major sub-lineages corresponding to ECs were estimated to be 50–150 years old.This study demonstrated the urgent need to monitorL monocytogenesstrains at the global level,and provided the unified approach needed for global harmonization ofL monocytogenesgenome-based typing and population biology.Hyden et al.[265]performed WGS-based core genome MLST analysis for serogroup determination of a panel of 172L.monocytogenesisolates spanning all 12 serotypes and 45 isolates with uncharacterized serotypes.They selected isolates with known serotypes and lineages to cover the entire genomic diversity of the species described by Ruppitsch et al.[262].These isolates could be clustered into the 2a,2c,4b or 2b clusters,according to their serotypes,respectively,by minimum spanning tree and neighbor joining tree data analysis,demonstrating the power of the new approach.

16S rRNA gene sequencing has been used for identification of bacteria at the species level[267].It is a multicopy housekeeping gene containing the highly conservative regions and hypervariable regions with considerable sequence diversity among different bacteria.Species-specific sequences within a given hypervariable region can be useful targets for diagnostic assays[268].Based on these characteristics,the partial(e.g.500bp)and the full(1500bp)of 16S rRNA gene can be amplified by PCR with appropriate primers and PCR amplified 16S rRNA gene fragments can be cloned and sequenced using Sanger dideoxy sequencing.Using 16S rRNA partial gene sequencing,two novel strains ofL monocytogeneswere identified[269]and a custom 16S rRNA library was established for differentiation and molecular subtyping ofListeriaspecies[270].

6.Conclusions and prospective

In this manuscript,we have comprehensively reviewed currently available methodologies for detection,characterization and subtyping ofL.monocytogenesin foods and environmental sources.These methodologies are developed according to different properties ofL.monocytogenes,including(a)unique colony morphology with macroscopic observation of bacterial growth on culture media;(b)metabolism activities and biochemical properties;(c)analysis of bacterial proteins/antigens and proteome and(d)species-specific genomic(gene)sequences.Each type of these methodologies has its own advantages and limitations/disadvantages,but none of them is the perfect one.Appropriate and different combinations of these methodologies with PCR-based methodologies can be applied in detection,characterization and subtyping ofL.monocytogenesaccording to particular need,availability of facilities/reagents/equipment and specific situation,which can be mutually supplementary and/or synergistic in overcoming limitations/disadvantages.Theoretically,the ideal methodologies should be rapid,simple,specific,sensitive,accurate,and reproducible,and can be used to easily detect low number ofL.monocytogenesfrom complex foods and environmental sources.They should also have the ability to completely distinguish the dead from viable cells,be cost-effective without being inhibited by food matrices,can be precisely validated for common and universal applications,and operated by personnel without highly technical training.For the prospective,in addition to further improving these methodologies for overcoming their limitations/disadvantages,new approaches are needed to be developed and comprehensively validated.In this regard,applications of bacteriophage in detection and subtyping ofL.monocytogenes[271–274]represent a great potential and a promising direction.

Conflicts of interest

None.

Disclaimer

Some reagents and other research tools described here are available from commercial sources.We are not recommending a particular product,but only providing an example of where it might be available.The authors have no conflict of interest with the companies mentioned in this review.

Acknowledgements

Due to limited space we apologize to our colleagues that their invaluable works could not be included in this publication.Research described here was supported in part by Oak Ridge Institute for Science and Education,USA,and by the FDA Intramural Chief Scientist Challenge grant.